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Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13 000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid, suggesting that these promoters were targeted in a non-random fashion, and probably occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-κB(p50), NF-κB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCζ, EGFR, Ras family Abbreviations :B H 4, tetrahydrobiopterin; BNIP3, Bcl2/adenovirus EIB 19 kD-interacting protein 3; CAMK2D, calcium/calmodulin-dependent protein kinase II delta; DHAP, dihydroxyacetone phosphate; DOC, deoxycholate; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; GAP, glyceraldehyde 3-phosphate; GC, guanylate cyclase; Grp78, 78 kD-glucoseregulated protein; IAP, inhibitor of apoptosis protein; IEF, isoelectric focusing; IKK-β ,I κB-kinase-β ;I P 3, inositol triphosphate; MALDI-MS, matrix assisted laser desorption ionization mass spectroscopy; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKK, MEK kinase; NIK, NF-κBinducing kinase; NLS, nuclear localization signal; NaDOC, sodium deoxycholate; NO, nitric oxide; NOS2, inducible NO synthase; ONOO–, peroxynitrite; PDTC, pyrrolidine dithiocarbamate; PKCζ, protein kinase C-zeta; PKG, cGMP-activated protein kinase; PN-1, protease nexin-1; QDPR, quinoid dihydropteridine reductase; SERPIN, serine protease inhibitor; TEM, transmission electron microscopy; TPI, triose phosphate isomerase; Trx, thioredoxin; TPx, Trx peroxidase (peroxiredoxin); TR, Trx reductase; TRAF, tumor necrosis factor receptor-associated factor; TTFA, thenoyl trifluoroacetone.

Functional inactivation of BRCA1 is an important mechanism involved in breast cancer pathogenesis. Mutation is often responsible for BRCA1 inactivation in familial breast cancer, but is not responsible for the decreased levels of BRCA1 seen in a subset of sporadic breast cancer patients. To determine if aberrant cytosine methylation of the BRCA1 promoter is associated with decreased BRCA1 gene expression in human breast cancer, high resolution bisulfite sequence analysis was used to analyze the cytosine methylation status of the BRCA1 promoter in 21 axillary node negative breast cancer patients with known levels of BRCA1 expression. Aberrant cytosine methylation of the BRCA1 promoter was detected in three of 21 patient specimens. These three specimens also expressed the lowest levels of BRCA1. Results from this analysis show that aberrant cytosine methylation of the BRCA1 promoter is directly correlated with decreased levels of BRCA1 expression in human breast cancer, and suggest that epigenetic silencing may be one mechanism of transcriptional inactivation of BRCA1 in sporadic mammary carcinogenesis.

An attempt was made to characterize the genetic regulation of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) in the absence of the cloned gene. Four human cell lines, differing in AGT activity from very proficient to essentially absent, were assayed for gene amplification as a possible mediator of the methylation repair phenotype (Mer+, AGT activity and MER-, no AGT activity) using in-gel DNA renaturation and G-banded karyotype analysis. The former technique allows subsequent analysis of amplification units and cloning of observed amplified DNA fragments, a hopeful approach to the isolation of the human AGT gene. Within the sensitivities of the techniques, no correlation between AGT activity and gene amplification was observed in the four cell lines tested.

The present study was initiated to determine if DNA damage induced by the bifunctional anti-tumor alkylating agents melphalan, nitrogen mustard, a spontaneously activated derivative of cyclophosphamide or chlorambucil inhibits transcription in vitro, and to determine if the potential sites of transcription termination correlate with the sites of N7 guanine adducts predominantly formed by these agents. To assess drug effects on in vitro transcription, linearized plasmid DNA containing the 420-bp PstI fragment of exon two of the human c-myc oncogene was incubated with various concentrations of the drugs. After drug removal and further drug-free incubation, the sense strand of the c-myc insert was transcribed with either of two bacteriophage RNA polymerases in the presence of [32P]UTP. The labeled products of the reaction were electrophoresed next to the labeled products of RNA sequencing reactions, and the location of transcription termination along the DNA template was determined. The sites of transcription termination were then compared with the sites of drug-induced guanine N7 alkylation in the template, as determined by modified Maxam-Gilbert sequencing. At the drug exposures examined, all the drugs were shown to alkylate any guanine in the template. Transcription of this alkylated DNA, however, resulted in RNA molecules truncated not at every alkylated guanine, but at various discrete sites throughout the template. Transcription was terminated at every adenine pair examined in the melphalan-treated template, at selected guanine pairs in the nitrogen-mustard-treated template, and at selected adenine-guanine and guanine-adenine pairs in the chlorambucil-treated template. Transcription of cyclophosphamide-derivative-treated DNA was unaffected. These results suggest that only some bifunctional alkylating agents induce DNA damage capable of terminating transcription in vitro, and that these agents do so in a sequence-specific, drug-specific manner inconsistent with patterns of guanine N7 alkylation.